The long-term objectives of the proposal are to identify how RC3 (neurogranin) and related PKC substrates modulate second messenger pathways coupled to 7- transmembrane receptors in the mammalian forebrain. RC3 is a neuron- specific substrate of protein kinase C (PCK) that accumulates near excitatory asymmetric synaptic juctions in dendritic spines of primarily forebrain neurons. The central hypothesis for the project is that PKC- phosphorylated RC3 selectively modulates IP3/DAG second messenger pathways coupled to postsynaptic 7-transmembrane receptors, particularly metabotropic glutamate receptors. In Specific Aim 1, wildtype or mutant forms of RC3 will be co-expressed with the 5HT1C receptor in Xenopus oocytes and tested for their relative effectiveness in enhancing Ca2+ - activated Cl- currents evoked by serotonin. These studies will identify specific amino acid residues in RC3 that mediate its modulation of the IP3/DAG second messenger pathway. In Specific Aim 2, RC3's potential interaction with either Go, PLC or the IP3 receptor will be investigated in functional assays in Xenopus oocytes by measuring the sensitivity of phorbol ester-evoked and serotonin-evoked Cl- currents to selective drug inhibitors. Potential RC3 modulation of either GTPgammaS binding and ADP-ribosylation of Go protein, phospholipase Cbeta activity, or binding to the IPc receptor will also be assayed biochemically with purified rat brain fractions incubated with a RC3-78 synthetic peptide. In Specific Aim 3, RC3 functions in IP3-related signal transduction in neurons will be addressed in three different kinds of tissue culture experiments.